Introduction: : Female reproductive aging is associated with ovarian functional decline, ultimately leading to infertility. The extracellular matrix (ECM) plays a critical role in supporting ovarian function, specifically impacting follicle growth and oocyte quality. ECM disruption is a hallmark of ovarian aging, yet how and which ECM properties cause ovarian functional decline remains poorly understood.
Materials and
Methods: : To investigate changes in global ECM composition as a function of age, young (6 weeks) and aged (14-15 month) murine ovaries were harvested, enriched for ECM, and subsequently analyzed using mass spectrometry. Regional characterization of ECM changes was carried out using immunohistochemistry staining on ovarian tissues for identified ECM markers. In order to correlate these biochemical alterations to the biophysical changes, ovarian slices were stained with a fluorescent live collagen reporter, followed by spatial nanoindentation of the stroma. To examine how stiffness influences follicle growth and oocyte quality, young follicles were cultured in soft and stiff hydrogels, mimicking young and aged ovarian stromal stiffness, respectively. Follicle growth and oocyte quality were evaluated. RNA sequencing (RNA-seq) of follicular granulosa cells was performed to assess transcriptional changes as a function of age and stiffness. Candidate signaling pathways were perturbed using drug targeting on in vitro cultured follicles to rescue age- and stiffness-related oocyte dysfunction.
Results, Conclusions, and Discussions:: Here, we describe spatiotemporal changes in the ovarian ECM through a fully integrated approach that combines using bulk mass spectrometry, immunohistochemistry (IHC), and nanoindentation. Stromal matrix remodeling with age led to a significant increase in stiffness. To understand how this increase in stromal stiffness affects age-related follicular dysfunction, isolated young follicles were cultured in soft and stiff hydrogels mimicking young and aged ovarian stromal stiffness, respectively. Higher stiffness led to a decrease in follicle growth, oocyte quality. RNA-seq of cultured granulosa cells uncovered differentially regulated mechanosignaling pathways in stiff vs. soft hydrogels. Targeting specific highlighted pathways found candidate drug treatment rescued oocyte quality in stiff environments. Furthermore, this treatment improved aged oocyte quality in vitro. Taken together, our work provides mechanistic insight into how ECM mechanics contributes to ovarian dysfunction, providing potential therapeutic targets to tackle age-related infertility
